Patterns of protein adsorption in chromatographic particles visualized by optical microscopy

Area of Science:

• Chromatography
• Biophysical Chemistry
• Materials Science

Background:

• Understanding protein adsorption kinetics is vital for chromatographic process optimization.
• Current methods often lack the resolution to observe transient adsorption patterns at the single-particle level.
• Strong binding conditions present unique challenges for studying mass transfer dynamics.

Purpose of the Study:

• To develop and validate a new optical microscopy method for imaging transient protein adsorption patterns within individual chromatographic particles.
• To investigate the mechanism of protein mass transfer under strong binding conditions.
• To enable precise quantification of intraparticle diffusivity.

Main Methods:

• Utilizing the refractive index difference between protein-adsorbent and protein-free matrix.
• Employing white light microscopy to visualize the moving adsorption front as a bright ring.
• Applying the shrinking core model for quantitative analysis of experimental data.

Main Results:

• Successfully imaged transient protein adsorption fronts in real-time within spherical chromatographic particles.
• Observed shell-progressive diffusion mechanism for lysozyme and albumin adsorption onto an agarose-based cation exchanger.
• Demonstrated accurate and precise determination of intraparticle diffusivity as a function of protein concentration and mobile phase composition.

Conclusions:

• The developed microscopy method provides unprecedented insight into protein adsorption kinetics at the single-particle level.
• The findings confirm a shell-progressive diffusion mechanism, informing chromatographic model development.
• This technique offers a powerful tool for characterizing and optimizing chromatographic materials and processes.